Sandia researchers have created “zombie” mammalian cells that may function better after they die.

The simple technique uses a silica solution to coat a cell’s insides to form a near-perfect replica of its internal structure. The process opens the door to simplifying a wide variety of commercial fabrication processes from the nano- to macroscale.

The work, reported in a fall issue of the Proceedings of the National Academy of Sciences (PNAS), uses the nanoscopic organelles and other tiny components of mammalian cells as fragile templates on which to deposit silica. The researchers then heat the cell to burn off its protein. The resultant hardened silica structures are faithful to the exterior and interior features of the formerly living cell, can survive greater pressures and temperatures than flesh ever could, and will function better for some uses than when they were alive, says lead researcher Bryan Kaehr (1815).

Letting nature do the work

“It's very challenging for researchers to build structures at the nanometer scale,” says Bryan, who came to Sandia as a Truman Fellow. "We can make particles and wires, but 3-D arbitrary structures haven’t been achieved yet. With this technique, we don't need to build those structures — nature does it for us. We only need to find cells that possess the machinery we want and copy it using our technique. And, using chemistry or surface patterning, we can program a group of cells to form whatever shape seems desirable.”

Says University of New Mexico professor and Sandia Fellow Jeff Brinker (1002), “The process faithfully replicates features from the nanoscale to macroscale in a robust, three-dimensionally stable form that resists shrinkage even upon heating to over 500C. The refractoriness of these delicate structures is amazing.”

Because a cell is populated by a vast range of proteins, lipids, and scaffolding, says Bryan, its interior is ready-made to serve as models for catalysts, funnels, absorbents, and other useful nanomachinery.

For example, he says, “Catalysts that evolve in cells are large molecules (enzymes) that have to be in the right shape for their chemistry to work. Because structure is important to their function, if we can stabilize a catalyst in the shape it evolved, that’s extremely valuable. Imagine stabilizing a genetically designed biocatalyst at its most optimal shape and then using it in a 200 degree Celsius reaction in which it otherwise would have no chance of surviving.” The hardened silica would stabilize and protect the still-present protein as it did its work.

In its simplest, most immediate use, says UNM post-doctoral student Jason Townson, silicification may be the simplest, best method of preserving the structure of organic materials for imaging. “Formerly, for internal preservation and subsequent imaging, a cell would be fixed in formaldehyde or some other preservative. But many of these methods are labor-intensive. This method is simple. The preserved cells will never get sloppy in decay. And when we cracked open the resulting structure, we were blown away by how well the cell was preserved, down to the minor groove of the cell’s DNA.”

Like a Madame Tussauds wax portrait

Heating the cell to still higher temperatures, greater than 400 C, evaporates the organic material of the cell — its protein — and leaves the silica in a kind of three-dimensional Madame Tussauds wax portrait of a formerly living being. The difference is that instead of modeling the face, say, of a famous criminal, the hardened silica-based cells display internal mineralized structures with intricate features ranging from nano- to millimeter-length scales.

Construction occurs like this: Take some free-floating mammalian cells, put them in a Petri dish, and add silicic acid.

Through the action of methanol, a byproduct of the acid, the cell’s lipid layers — the protective casings that keep the cell intact — are softened and made porous enough for the silica to flow in at about the temperature of the human body.

The silicic acid, for reasons still partially obscure, enters without clogging and in effect embalms every organelle in the cell from the micro- to the nanometer scale.

If the cell isn’t heated, the silica forms a kind of permeable armor around the protein of the living cell. This may support it enough to act as a catalyst at temperatures and pressures undreamed of by nature.

“Once we’ve stabilized the cellular structure, it can still carry out reactions and more important, that reaction is stable enough to work at high temperatures,” Bryan says. “It’s a means to take a soft, potentially valuable biological material and convert it to a fossil that will stay on our shelves indefinitely.”

Ordinarily, preserving something organic means freezing it, which is energy-intensive, he says. Instead, “We’re doing rapid fossilization: quickly converting a protoplasmic cell into a hard structure that will stand the test of time.”

The unusual but simple procedure may serve as a model for creating hardier classes of nanoscopic products.

Experiments showed the cell can be used as a reverse mold from which, at 900 C, a porous carbonized structure results from heating cell protein in a vacuum. (Put in ordinary terms, burning wood in air leaves a residue of structureless soot. The zombie heating method results in a high-quality carbon structure.) Subsequent dissolution of the underlying silica support decreased the cell’s electrical resistance by approximately 20 times. Such materials would have substantial utility in fuel cells, decontamination, and sensor technologies.

Going beyond large sponges

That such extraordinary results can be achieved by silicifying cells indicates, according to the technical paper, that many soft cellular architectures could be “feedstock for most materials processing procedures, including those requiring high temperatures and pressures.”

Other porous material structures, relying on titanium instead of silica, have been formed using the organic template technique. Other metal oxides, says Bryan, are a possibility. These would have advanced structural functions or could serve as catalysts.

The work follows the efforts of a number of scientific groups, including Bryan’s, that have built gel-like structures, copied them with silica, and then burnt off the gel to create, in effect, large sponges.

“Now we can change the biological shape and calcify (heat) it, so for the first time we get new irregular structures,” Bryan says.

In the PNAS paper, Bryan’s team used dissociated cell culture from various human, chicken, and mouse organs as starter material. Grown in flasks, these are soft, delicate objects that normally would eventually exist in a multicellular structure like a person (or a chicken or mouse).

“Since then we have found that the procedure can silicify an entire organism — in this case, a chicken embryo — which is somehow creepy,” says Bryan. “But it proves that larger-order animal forms (that is, humans) can, in principle, be glassified just like single cells using this technique.”

The work was supported by DOE’s Office of Science. Other authors of the technical paper are Jeff Brinker (1002 and UNM), Brian Swartzentruber (1131 and the Center for Integrated Nanotechnologies), Robin Kalinich (2501), and Darren Dunphy and student Yasmine Awad of the University of New Mexico.

Looking for nefarious intent in the cyberworld

With that in mind, Sandia researcher Jeremy Wendt (5632) is trying to figure out how to recognize potential targets of nefarious emails and put them on their guard.

He’s working to reduce the number of visitors that cyberanalysts have to check as possible bad guys among the tens of thousands who search Sandia websites each day.

His ultimate goal is to spot spearphishing. Phishing is sending an email to thousands of addresses in hopes a few will follow a link and, for example, fall for a scam offering millions of dollars to help a Nigerian prince wire money out of his country. Spearphishing, on the other hand, targets specific email addresses that have something the sender wants. “Spearphishing is scary because as long as you have people using computers, they might be fooled into opening something they shouldn’t,” Jeremy says. Even if an outsider gets into a Sandia machine that doesn’t have much information, that access makes it easier to get into another machine that may have something, he says.

Jeremy has been working on algorithms that separate web crawlers from people using browsers, and he has been able to split those groups. He believes the work to date will help security because it allows analysts to look at groups separately.

Identifying malicious intent

Cybersecurity’s Roger Suppona (9317) says the ability to identify the possible intent to send malicious content might enable security experts to raise a potential target’s awareness. “More importantly, we might be able to provide sufficient specifics that would be far more helpful in elevating awareness than would a generic admonition to be suspicious of incoming email or other messages,” he says.

Jeremy, in the final stretch of a two-year Early Career Laboratory Directed Research and Development grant, presented his work last year at a Sandia poster session.

He has been looking into behaviors of web crawlers vs. people browsing to see if that matches how computers identify themselves when asking for a webpage. A browser’s computer generally says it can interpret a particular version of HTML — HyperText Markup Language, the main language for displaying webpages — and often gives browser and operating system information. Crawlers identify themselves by program name and version number. A small number Jeremy calls “nulls” offer no identification, perhaps because the programmer omitted that information, perhaps because someone wants to hide.

What Jeremy is looking for is a computer that doesn’t identify itself or says it’s one thing but behaves like another and trolls websites in which the average visitor shows little interest.

Going to an Internet site creates a log of the search. Sandia traffic is about evenly divided between web crawlers and people browsing. Crawlers tend to go all over; browsers concentrate on one place, such as jobs.

Crawlers, also known as bots or robots, are automated and follow links like Google or Bing do. “When we get crawled by a Google bot, we aren’t being crawled by one visitor, we’re being crawled by several hundreds or thousands of different IP addresses,” Jeremy says. An IP or Internet Protocol address is a numerical label assigned to devices on a computer network, identifying the machine and its location.

Distinguishing bots from browsers

Jeremy wants to distinguish bots from browsers without having to trust they are who they say they are. He expects some are lying, so he looked for ways to measure behavior.

The first measurement deals with the fact bots try to index a website. When you type in search words, the web crawler looks for pages associated with those words, disregarding how they’re arranged on a page. That means a bot pulls down HTML files far more often than other things.

Jeremy first looked at HTML downloads. Bots should have a high percentage. Browsers pull down smaller percentages.

More than 90 percent of the nulls pulled down nothing but HTML — typical bot behavior.

A single measurement wasn’t enough, so Jeremy devised a second based on another marker of bot behavior: politeness.

Bots could suck down webpages from a server so fast it would shut down the server to anyone else, Jeremy says. That might prompt the site administrator to block them.

So bots take turns. “They say, ‘Hey, give me a page,’ then they may crawl a thousand other sites taking one page from each,” Jeremy says. “Or they might just sit there spinning their wheels for a second, waiting, and then they’ll say, ‘Hey, give me another page.’”

Browsers go after only one page but want all images, code, and layout files for it instantly. “I call that a burst,” Jeremy says. “A browser is bursty; a crawler is not bursty.” Bursts equal a certain number of visits within a certain number of seconds.

What ‘bursty’ behavior indicates

Ninety percent of declared bots had no bursts and none had a high burst ratio. Sixty percent of nulls also had no bursts, lending credence to Jeremy’s belief they’re bots.

But 40 percent showed some bursty behavior, making them hard to separate from browsers. However, normal browsers behave predictably. When Jeremy combined both metrics, most nulls fell outside those parameters.

That left browsers who behaved like bots. “Now, are all these people lying to me? No. There could be reasons somebody would fall into this category and still be a browser,” Jeremy says. “But it distinctly increases suspicions.”

So he also looked at IP addresses. Unlike physical addresses, IP addresses can change. Say you plug your laptop into the Internet at a coffee shop, which assigns you an IP address. After you leave, someone else shows up and gets the same IP address. So an IP address alone doesn’t necessarily distinguish users.

There’s another identifier: a particular browser on a particular operating system, which leads to what’s called a user agent string. There are thousands of distinct strings.

IP addresses and user agent strings can collide, but Jeremy says odds are dramatically lower that two people will collide on the same IP address and user agent string within a short period such as a day. That tells him they’re probably different people.

Now he needs to bridge the gap between splitting groups and identifying targets of ill-intentioned emails. He has submitted proposals to further his research after the current funding ends this spring.

“The problem is significant,” he says. “Humans are one of the best avenues for entering a secure network.”

High-stakes countdown at Kauai

In the Pacific Ocean, some sailors will be tested. A target missile launched from Sandia’s Kauai Test Facility (KTF) will fly across the sky and, if all goes according to plan, the Navy’s newest interceptor missile, the Standard Missile-3 Block IB, will shoot it down in an operation similar to defending the US from an offensive missile attack.

Past test operations have been described as hitting a bullet with a bullet.

Most people think of countdowns as the television voiceover saying, “10, 9, 8 . . . ,” but they are much more.

Days and weeks before the launch, Sandia employees arrive at KTF, a 132-acre launch site at the Pacific Missile Range Facility (PMRF) on the western tip of the Hawaiian island of Kauai, to support the test of the US Missile Defense Agency’s Aegis Ballistic Missile Defense Program.

At the PMRF gate, drivers are told to go “right at the stop sign and all the way down.” The remote spot is no accident: it has little interference from the radio frequencies found on the East and West coasts; there’s an expanse of ocean to work in and it’s near the Pacific Fleet, says Vince Salazar, senior manager of Sandia Missile & Air Defense (5410).

Test targets arrive at the Missile Assembly Building, which contains a 30-by-70-foot high bay with 10- and 20-ton cranes and office space, where they are assembled and tested, manager Steve Lautenschleger (5419) says.

Nearby, two white canvas “clamshells” on wheels protect two large rail launchers, which are used to launch both guided and ballistic missiles, from the weather, Steve says. Nearby are a vertical stool launcher, which will be used for this missile, and a universal rail launcher.

Across from the vertical launcher is the bunker-like Launch Operations Building (or LOB, pronounced el-oh-bee), the site’s communications hub, topped by 15- and 20-foot white dish antennas that will receive telemetry from the missile.

Reuben Martinez (5419), the test director for this mission, first came to KTF as the “computer guy.” He explains that thousands of data points will be analyzed and turned into graphic displays that help controllers quickly determine whether the test missile is flying along its intended flight path. They use that information in real time to make recommendations about whether to continue the mission. It’s also used after the flight for further analysis or making changes when things don’t go the way they should, he says.

Sandia employees and contractors begin their three practice countdowns with the arrival of Uncle Tom Takahashi, an 81-year-old elder (kapuna) from a local church who blesses the missile.

“Sandia, her name is,” he pauses, then, “Uilani,” which means beautiful heaven. The Navy missile is named “Keiki ale ale o kekai,” which means choppy water of the ocean, describing the condition of the Pacific in recent days.

The practice countdowns include: an internal readiness test, the first time everyone comes together for the mission; a dry run, to verify the countdown is correct; and a dress rehearsal, when the aircraft and ships participate so they understand the timing and the countdown must be finalized, says Margaret Scheffer (5419), the Sandia test officer. Margaret will communicate with PMRF, the lead range on launches from KTF. Reuben will be responsible for all the intrarange issues involving the missile, launch pad, and all the KTF launch assets.

“It’s high pressure in the terminal countdown, so it’s important to have these practice countdowns,” Reuben says.

Launch day

When employees arrived at KTF, the smell of smoke hung in the air from a wildfire on nearby Makaha Ridge.

Reuben arrives from PMRF’s Range Operations Control Center (ROCC, pronounced rock) and says the launch is a go.

Notices already have been sent to ships and aircraft, telling them to avoid the Notice to Mariners/Notice to Airmen (NOTMAR/NOTAM) area surrounding the potential debris field. On launch day, the area is again checked for “range foulers” that haven’t heeded earlier warnings, Margaret says.

Along the raised concrete platform that connects the white 40-foot trailers that house staff and customer offices, Margaret walks to the LOB. Is she is nervous ahead of the launch? “Not yet. I don’t get nervous until the last 30 minutes.”

During three practice countdowns, Margaret has demonstrated nerves of steel under pressure, seasoned by the dozens of countdowns she has run. She is an aeronautical engineer who worked on Sandia’s Strategic Target System (STARS) and other missiles.

Reuben and Margaret sit down behind a bank of video and computer monitors, keyholes, knobs, and switches on a raised platform in the center of the LOB to start the countdown. Three large video screens show the launch pad and the test missile’s sides, graphics of the flight path once the test missile is launched, and pictures from the rocket’s on-board camera.

A large digital clock with red numbers ticks away the hours at the left-hand side of the room.

T minus 06:00:00

“T minus six hours,” an automated voice says.

This is the countdown’s unceremonious start. The KTF team must complete more than 500 steps to launch the 42-foot-tall single-stage guided missile. Each step is listed on a spreadsheet that Reuben has tweaked to perfection in the late night hours during the three practice countdowns.

If the on-screen boxes containing each step turn yellow, Reuben and Margaret state the task hasn’t been completed within one minute of its scheduled time. After 60 seconds, the print turns red and remains so until the task is completed and checked.

T minus 05:33:03

One of the first major steps is to verify the launch pad and missile are ready for launch. The latter involves turning on and verifying you can receive telemetry from the equipment, and confirming that the systems are working.

After Margaret receives a series of numbers from PMRF, an expert in the field radios to say several numbers are unusually low.

Reuben considers the problem, leaning forward in his chair and then pacing. He asks whether one of the doors on the vertical launch tower hadn’t been opened properly and saturated the radio signal, but the response from the field was that the door was fine.

The wheels turn in his head as he ticks off what could be causing the odd readings: Are the PMRF antennas turned in the correct direction, he asks. Sure enough they were pointed the wrong way. Problem solved.

“You need to have situational awareness,” Reuben says. “When something doesn’t look good, you have to figure it out on the fly.”

T minus 05:00:00

“T minus five hours,” drones the automated voice.

Reuben’s and Margaret’s jobs are stressful and involve major multitasking. Imagine monitoring 15 communications networks, or loops, chattering all at once, having to problem-solve within seconds or risk delaying a launch, and working and communicating with multiple agencies to make the launches happen.

“It’s a stressful job, but it’s exciting,” says Reuben, who has been test director for five launches and recently also became KTF’s site manager. “Margaret and I are a good team. We’ll get in a groove, and I know what she’s going to do and she knows what I’m going to do.”

As they work, many times a single utterance or a nod indicates who’s going to handle which task and they often cover for each other like clockwork.

Employees then verify that the Flight Termination System (FTS) works. This missile contains an FTS, which could be triggered remotely to destroy the vehicle if it flies off course before it hits something it shouldn’t, Steve says.

A large white weather balloon is tethered to the 80-foot tall launch tower to provide a PMRF team a visual on the vehicle pad location. This gives the PMRF team a line of sight to verify how the rocket leaves the launch pad in the brief period before it’s high enough to be detected by radar, Margaret says.

The balloon is the same as the five or six weather balloons Sandia contractors at KTF launch to 100,000 feet to measure high altitude weather conditions, such as the jet stream, say mechanic Charlie Vegas and groundskeeper Michael Mier (both 5419-1).

“In the beginning, we didn’t get involved with the launches. Now we’ve gotten involved with pretty much everything they do,” Charlie says.

Ken Dama (5419-1) agrees that the local contractor staff is an integral part of KTF, in day-to-day operations and mission support.

Today, the weather balloon above the launch pad accidentally gets wrapped around a lightning tower and partially deflates. Steve is concerned that the balloon could interfere with the launch tower being pulled away from the missile.

“There’s always some kind of unplanned event,” Reuben says. During a practice countdown, four chickens scurried across the launch pad and pigs once ran across the launch pad during an actual countdown.

Steve decides the balloon must be cut down, and confers with Wayne Itokazu (5419), who is responsible for the weather balloon launches and data acquisition.

“I like these types of crises better than when a target missile doesn’t work,” Steve says. “This is not so bad.”

Should the report be negative, the launch could be scrapped, Reuben says.

While the support Terry gives from Sandia is just one example, Vince says Sandia’s involvement in KTF pre- and post-launch and during the mission operations is key to KTF’s success, particularly when Sandia-developed rockets and payloads are launched.

“Sandia is what makes KTF unique. It is Sandia doing the technical work, developing the target missile systems and also having major responsibility for the payloads that were out there,” Vince says. “You get to see the countdown and liftoff of these experiments. It is sealing the deal.”

Suddenly, 13 steps on Reuben’s and Margaret’s screens turn red. Employees are on the launch pad arming the missile, but another group needs to start its work.

Calls are coming in from the ROCC asking when the next task will start, as the entire screen goes red. The tension in the room rises slightly, as Steve and Margaret discuss how to keep the launch moving forward, but about an hour later PMRF puts a planned two-hour hold into effect, KTF suddenly catches up, and faces show relief all around.

With the missile now prepared for launch, all non-essential personnel are cleared out of KTF, in case of an accident, to watch the launch from a nearby field.

Employees take the arm plug, a red canister about the size of an egg, and remove the green safe plug to make the connections on the launch pad necessary for the launch, a task simulated during the practice runs. The plugs are kept safely in a lock box behind the central operations desk.

There are about 22,000 pounds of explosives on the target missile, so safety is paramount, Steve says.

T minus 00:15:00

With about 15 minutes to go, Steve and Reuben say the LOB becomes very still as everyone focuses on their jobs.

“No one’s talking about their weekend or cracking jokes,” Reuben says. “When you get to the 10-minute mark, you think, ‘This is serious. We’re probably going to go. We’re not turning back now.’”

T minus 00:05:00

“The three-word hold is in effect,” comes over the radio. Reuben explains that anyone who sees any reason to hold the launch can say “hold, hold, hold” into the radio and everything is halted. During the dress rehearsal, two holds were called outside KTF as last-minute checks were made, but so far the real launch has had none.

A set of keys used to initiate the launch were handed out to controllers early in the countdown. The keys are turned after each person’s steps are completed, so along the way, anyone can delay turning their key to hold the launch. There’s also a big black “HOLD” button on the test director’s display.

“If we’re going to hold, this is when it’s going to happen,” Reuben says.

Margaret adds: “There are so many ways to hold and only one way to launch.”

During the last minute, Reuben explains a day later, his heart is pounding. He takes his hand off his mouse so he doesn’t accidentally click a step before it’s complete.

An automated voice indicates 90 seconds, then 60 seconds to go.

A display in the corner of Reuben’s monitor shows six of seven mandatory interlocks for launch are green, then the last one is checked by Booster Control, Rod Stanopiewicz (5419), with 17 seconds to go. Voices from the communications networks are heard repeating that the launch is a go.

And with an orange blaze the target missile moves slowly off the launch stool and into the night sky.

In a field about two miles away, a crowd of Sandia employees and contractors, Orbital Sciences staff (Orbital Sciences supplied the missile), and the military waits in the dark.

From behind a line of trees a whitish-orange glow lights up a half circle of the dark sky. Silently a brightly glowing orange oval rises slowly above the trees. Then the sound hits, first a rumble, followed by a metallic roar that lasts for nearly a minute as the oval becomes smaller and smaller, until it is a tiny orange ember among the stars.

A small cheer and a sense of relief washes over the LOB, Reuben says.

The target missile transmits telemetry to KTF, where Ed Mader (5419) is splitting his time between operating the tracking antennas and monitoring the computer room with Steve Sanchez (5419). Wes Crownover (5419), who records all data, but uses Best Source Selector equipment to determine the data with the least errors caused by the transmission and routes it to Sandia and Orbital Sciences.

Across the LOB, Larry Young (5422) generates displays on computers that translate telemetry within milliseconds into graphs on 17 monitors that show whether the target missile is flying along its intended path, viewed as a green line within an orange corridor. Larry says the guided missile should fly along the green line. Should the missile move outside the orange corridor, the Navy would abort its launch to avoid wasting a missile.

Sitting yards away from Larry is Tom Johnson of the Johns Hopkins University/Applied Physics Laboratory, who studies the graphs to tell PMRF whether it’s a good target.

On this test, Johnson has a 15-second window to make the call, which he says is a “leisurely pace,” compared to as little as 5 seconds he sometimes has on other flights. Today, the rocket flies straight.

The voice of Eric Hedlund, test director of the US Missile Defense Agency’s Aegis Ballistic Missile Defense (BMD) Program, is heard from the ROCC: “Good target, good target.”

“Confirm,” comes the answer, which means the Navy can try to intercept the incoming missile.

“Eagle away!” The Navy is ready to launch its interceptor. All eyes are on the front video screen, watching as two yellow lines grow longer and longer and intercept at a yellow dot.

In the field, the missile sparks orange as the interceptor flying from the Navy ship in the Pacific Ocean hits it. An excited voice says “Mark India!” on the radio.

“Whoo hoo!” People shout through the dark. “Congratulations!”

Back in the LOB, the control desk is quiet because it’s unclear whether the interceptor hit its target, Steve says. But Wes, the telemetry expert, sees his data go dark, so he and a few others know the mission is successful. Their joy quickly spreads across the room.

One contractor exits the LOB with a huge smile across his face.

Steve looks happy and relaxed. The launch was “smooth … very smooth” and no holds were called in the final minutes.

“I really like this job. You really feel like you’re doing something important,” Reuben says. “I bet the president knows we did this tonight.”

Margaret adds: “You feel instantly excited that it’s over. Then you can breathe. Then you start thinking about starting work on the next one.”

Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.